A thorough understanding of the molecular machinery that operates in nociceptive sensory neurons will lead to greater understanding of the protective molecular mechanisms of acute pain. To identify nociception molecules, we previously carried out a genetic screen for nociception defective mutants using the fruitfly Drosophila melanogaster. This led to our discovery of the painless gene, which shows enriched expression in nociceptive sensory neurons and is required for acute nociception. The painless gene, as well as a related gene named dTRPA1, encode homologues of TRPA1. Human mutations in TRPA1 mutations lead to familial episodic pain syndrome (FEPS) and TRPA1 is highly expressed in nociceptor neurons. We have identified pickpocket as a critical regulator of mechanical nociception which is specifically expressed in Drosophila nociceptors. The purpose of this proposal is to identify other genes expressed in nociceptor neurons of Drosophila and to test the functional requirement of those genes in nociception. Building upon our recent studies that identified the Class IV multidendritic neurons as polymodal nociceptors of the Drosophila larva we will: 1) Determine the complete transcriptome of nociceptive Class IV multidendritic neurons. 2) Test the functional requirement of genes with enriched expression in nociceptive sensory neurons using tissue specific RNAi knockdown in these cells in vivo. 3.) Functionally characterize a subset of novel genes that we identify to be essential for nociception in Aims 1 and 2. In the short term, these studies will provide critical insight into molecular and cellular mechanisms of nociception using a high throughput approach that is made possible using Drosophila. In the long term, these studies may eventually allow for the identification of evolutionarily conserved mechanisms that contribute to human nociception. ! PUBLIC HEALTH RELEVANCE: The new nociception signaling molecules that we have and will discover in the proposed studies will have a sustained and profound impact on the field of nociception research.